Acute myeloid leukemia cells constitutively express the immunoregulatory enzyme indoleamine 2,3-dioxygenase.
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Acute Disease↗
Biomedical subjects
A L Horenstein
Publications and source records attributed to A L Horenstein.
Monoclonal antibodies and therapy of human cancers.
This survey is an overview of the applications of murine, humanized and recombinant monoclonal antibodies for in vivo diagnostic and therapeutic applications. Monoclonal antibodies (mAb) have been applied to the diagnosis and therapy of an array of human diseases. The initial failures of early clinical trials have been overcome through the production of a new generation of mAb which features reduced immunogenicity and improved targeting abilities. The early models of mAb therapy were focused on enhancing the cytolytic mechanisms against the tumor cells. More recently, successful mAb-based therapies were targeted to molecules involved in the regulation of growth of cancer cells. This has highlighted the relevance of understanding receptor-mediated signaling events, and may provide new opportunities for anti-tumor antibody targeting. Despite all the difficulties, clinical data is outlining an increasingly significant role for antibody-mediated cancer therapy as a versatile and powerful instrument in cancer treatment. One reasonable expectation is that treatment at an earlier stage in the disease process or in minimal residual disease may be more advantageous.
Journal Article↗
Identification of a 220-kDa membrane tumor-associated antigen by human anti-UK114 monoclonal antibodies selected from the immunoglobulin repertoire of a cancer patient.
Human monoclonal antibodies (HuMAb) specific for a 14-kDa perchloric acid-soluble protein (defined as UK114) were produced by somatic fusion of the human-mouse myeloma K6H6/B5 with Epstein-Barr virus-transformed peripheral B lymphocytes from a cancer patient previously treated with UK101 preparations, containing the UK114 protein. Three IgM-secreting clones were selected on the criteria of specificity for the purified UK114 protein immobilized onto plastic and adapted to grow in a serum-free medium. The reactivity of these antibodies showed a broad distribution pattern restricted to fresh tumor tissues and tumor cell lines, mainly of the adenocarcinoma type. None of the normal cells, nonmalignant cell lines, and normal tissues surrounding the neoplastic lesions were reactive. The immunochemical analysis of the target antigens showed that the HuMAb recognize a molecule of 220 kDa selectively expressed by the surface of tumor cells, as well as a cytoplasmic 14-kDa protein. The 220-kDa antigen was different from other tumor-associated antigens with similar molecular mass and, so far, unique. In the presence of human complement, two of three HuMAb are cytotoxic for tumor cells expressing the 220-kDa surface antigen. The tumor specificity and the lytic ability attributed to these HuMAb are promising features for the exploration of future clinical applications.
Animals↗
c-kit is expressed in soft tissue sarcoma of neuroectodermic origin and its ligand prevents apoptosis of neoplastic cells.
During development, mice with mutations of stem cell factor (SCF) or its receptor c-kit exhibit defects in melanogenesis, as well as hematopoiesis and gonadogenesis. Consequently, accumulating evidence suggests that the c-kit/SCF system plays a crucial role in all of these processes and in tumors which derive from them. Especially in neuroblastoma (infant tumors of neuroectoderm crest derivation such as melanocytes) it would appear that an autocrine loop exists between c-kit and SCF, and that the functional block of the c-kit receptors with monoclonal antibodies (MoAbs) results in a significant decrease in cellular proliferation. We studied the expression and role of c-kit and SCF in cell lines of soft tissue sarcoma of neuroectodermic origin, such as Ewing's sarcoma (ES) and peripheral neuro-ectodermal tumors (PNET). Using flow cytometry with MoAb CD117 PE, c-kit expression was highlighted in all six of the cell lines examined. This receptor was specifically and functionally activated by SCF, as shown by the binding experiments and the intracellular phosphotyrosine and immunoprecipitation studies that were performed. Using reverse transcriptase polymerase chain reaction analysis, five of the six cellular lines expressed the mRNA of SCF. In the medium measured by using an enzyme- linked immunosorbent assay, low concentrations of SCF were found: only the TC32 cellular line produced significantly higher levels (32 pg) than control. In serum-free culture the addition of SCF reduced the percentage of apoptotic cells from 25% to 90% in five out of the six cellular lines. This observation was confirmed by (1) the functional block of c-kit with MoAb: after 7 days of culture more than 30% of the cells were apoptotic (range 31.5% to 100%) in five out of six cell lines and there was also a decrease in the percentage of cells in phase S, and (2) c-kit antisense oligonucleotides: in the cellular lines treated with oligonucleotides (in relation to the untreated lines) there was a notable reduction (P < .001) both in the absolute number of cells and the 3H-thymidine uptake. These results indicate that ES and PNET express c-kit and its ligand SCF and that SCF is capable of protecting the tumor cells against apoptosis. Furthermore, the reverse transcriptase-polymerase chain reaction performed on the biopsies revealed the presence of mRNA both of SCF and c-kit in practically all of the samples studied. Our in vitro data lead us to assume that SCF may also inhibit tumor cell apoptosis in vivo.
Animals↗
CD38 binding to human myeloid cells is mediated by mouse and human CD31.
Soluble forms of membrane receptors are emerging candidates as physiological regulators of leukocyte trafficking. In the present study, we found that the soluble form of the CD38 antigen (sCD38) bears a binding domain of low affinity for a cellular receptor on U937 cells. Cross-linking and peptide-mapping studies confirmed the physical association and the identification of the U937 receptor as a 130 kDa protein. The binding of sCD38 to the receptor was differentially inhibited by several monoclonal antibodies against the CD31 cell-adhesion molecule. Thus the interaction was analysed through direct association of soluble and membrane CD38 with soluble recombinant murine CD31 with three N-terminal and with all six extracellular Ig domains. Cross-linking experiments on U937 intact cells, and ligand blot assays of the immunoprecipitated CD38 molecule, indicated that (i) the recognized epitope is determined by the tertiary structure of the molecule, and that (ii) the binding domain involved resides in the ectocellular portion of the CD31 molecule, more precisely in the first three N-terminal domains. A comparative functional activity between murine and human CD31 was also explored. The data presented suggest that (i) human CD31 bears a highly functional similarity with its murine counterpart, as it is a receptor in myeloid cells with more than one ligand (the alphavbeta3 integrin and the CD38 molecule), and that (ii) the activity of sCD38 as decoy molecule for CD31 may play an important role in cell-cell interactions in physiological and pathological conditions.
ADP-ribosyl Cyclase↗
Human CD38 ligand. A 120-KDA protein predominantly expressed on endothelial cells.
Human CD38, a pleiotropic molecule with ADP-ribosyl cyclase activity, regulates activation and growth of several cell types. Its in vivo function is incompletely determined, mainly due to the lack of evidence concerning the existence of a single or multiple ligands. We recently observed that CD38 rules a selectin-type adhesion between lymphoid cells and HUVECs. A panel of murine mAbs raised against HUVEC included one (Moon-1) constantly blocking the CD38-mediated adhesion of several cell lines to HUVEC. Tissue distribution studies and an extended immunohistochemical analysis on the majority of normal human tissues revealed that the Moon-1 molecule displays a unique pattern of expression, being present at high levels on resting and activated vascular endothelium, on the majority of monocytes, platelets, NK cells, and to a lesser extent on T, B, and myeloid cells. The Moon-1 structure of an apparent molecular mass of 120 kDa proved to be a ligand for human CD38, as inferred by the direct binding observed when using a chimeric mouse CD8 alpha-human CD38 (mCD8 alpha-hCD38) molecule as a probe in Western blot experiments. Furthermore, Ab-induced modulation experiments highlighted an association between the Moon-1 molecule and human CD38 on the surface of cell lines coexpressing the two structures, which also share a common regulation system of surface expression. Finally, direct ligation of Moon-1 on T cell lines caused a relevant increase in the cytoplasmic concentration of calcium ([Ca2+]i).
ADP-ribosyl Cyclase↗
Identification and characterization of an active soluble form of human CD38 in normal and pathological fluids.
Human CD38 is a transmembrane glycoprotein involved in lymphocyte activation and adhesion to endothelium. The ectocellular domain of the molecule possesses properties of a bifunctional enzyme catalyzing both the synthesis from NAD+ and the hydrolysis of the calcium-releasing metabolite cyclic ADP-ribose (cADPR). Surface expression of CD38 (mCD38) is rapidly and almost completely down-modulated upon ligation by specific mAb in cells from different lineages. The data presented here also show that, in addition to the existence of a mCD38, a soluble form of CD38 (sCD38) is detectable in the cell culture supernatant of allo-activated T lymphocytes and of several tumor cell lines. sCD38 is also present in vivo and is assayable in normal (fetal serum and amniotic fluid) and pathological (serum and ascites from patients with multiple myeloma, and serum from patients with AIDS) biological fluids. Immunoaffinity chromatography, SDS-PAGE and Western blot analyses with mAb and polyclonal antibodies, along with metabolic labeling, yield a body of data concerning the structure of sCD38, which displays a M(r) of 39 kDa. Native sCD38 maintains the ability to inhibit the binding activity of different anti-CD38 mAb and still catalyzes the synthesis and the hydrolysis of cADPR at the same ratio observed with mCD38. Furthermore, cross-linking experiments indicate that the purified soluble molecule binds a 120 kDa molecule expressed by monocytoid cells and identified as a candidate ligand for human mCD38.
ADP-ribosyl Cyclase↗
99mTc direct labeling of anti-CEA monoclonal antibodies: quality control and preclinical studies.
The anti-carcinoembryonic B2C114 monoclonal antibody was radiolabeled with 99mTc by a direct method and quality control tested in vitro by instant thin layer chromatography, gel column scanning and cellulose acetate electrophoresis and assessed in vivo for radioimmunodetection on a murine spontaneous mammary carcinoma. The optimal results of percent 99mTc bound to protein were obtained at a dithiothreitol:antibody molar ratio ranging from 800:1 to 1000:1 and at a methylene diphosphonate:stannous fluoride weight ratio of 4.3:1. Although cysteine removed up to 18% of the label during the first 4 h, the stability of the tracer appeared to be excellent in human serum at 37 degrees C and when challenged with DTPA. 99mTc-labeled B2C114 demonstrated good and specific in vivo tumor targeting.
Animals↗
Direct labeling of monoclonal antibodies with 99mTc and radioimmunodetection of a murine mammary carcinoma with 99mTc-B2C114.
Two anti-CEA antibodies, B2C114 and IORCEA1, were radiolabeled with 99mTc by two direct methods (mercaptoethanol and ascorbic acid reduction), and the radio-immunoimaging properties of B2C114 were assessed in mice bearing an M3-reactive tumor. The labeling efficiency was greater than 90% as measured by ITLC in saline, methylethylketone and with serum albumin impregnated sheets using ethanol: water: NH4OH (2:5:1). The label was stable to challenge with excess DTPA, and in the case of ascorbic acid reduction, serum analysis showed that 10-15% of the radioactivity was lost during incubation. In vitro studies demonstrated that the radiolabeled antibodies retained their immunoreactivity. Biodistribution studies in normal Balb/c mice showed that the pattern of uptake was quite similar for both antibodies. Biodistribution of the 99mTc-B2C114 and image studies in the animal model showed that the tumor was clearly visualized and that B2C114 labeled with 99mTc is a possible candidate for human radioimmunodetection of CEA-expressing tumors.
Animals↗
Real-time kinetic analysis applied to the production of bispecific monoclonal antibodies for radioimmunodetection of cancer.
An automated biosensor system designed for measuring molecular interactions in real-time and without labelling of the reactants has been used to evaluate the association/dissociation rate and affinity constants of bivalent monoclonal antibodies and a monovalent bispecific monoclonal antibody. Observed differences in affinity between parental and bispecific antibody produced were related to the association rate constants, since the dissociation rate constants were in the same range. Values were also closely related to radioimmunochemical data. These results indicate that the biosensor system, besides presenting several advantages for characterizing antigen-antibody interaction, is valuable for selecting monoclonal antibodies with properties which might be useful in the development of bispecific monoclonal antibodies.
Antibodies, Bispecific↗
Specific biodetection of a murine spontaneous mammary carcinoma with labelled anti-human blood group-A monoclonal antibody.
The expression of cell surface antigens of the spontaneous transplantable M3-murine tumour was studied by means of the monoclonal antibody (MAb) B2C114 which recognizes the human blood group-A carbohydrate antigen. Following radioiodination the MAb retained their immunoreactivity and demonstrated a significantly higher in vitro binding with isolated M3-tumour cells as compared with a control antibody. B2C114 revealed 10(6) antigenic sites per cell, with a constant affinity of 5.1 x 10(9)/M. Biodistribution studies showed that B2C114 discriminated between malignant tumour and mouse normal tissues. Radioimmunodetection of Balb/c mice bearing s.c. M3-tumour showed that tumour was specifically defined without subtraction 1 day after injection of the radiolabelled antibody.
ABO Blood-Group System↗
[Monoclonal antibodies in the in-vivo diagnosis of cancer].
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Antibodies, Monoclonal↗
An improved selection procedure for the rescue of hybridomas. A comparative study of methotrexate versus aminopterin.
Several of aminopterin's drawbacks such as photosensitivity and high toxicity prompted us to compare the ability of methotrexate to select for hybridomas. Assessing the effect of both drugs on X63/Ag 8.653 myeloma cells and hybrid cells secreting monoclonal antibodies by tritiated thymidine incorporation and percentage of viable cells, we have shown that methotrexate could be used in place of aminopterin for the rescue of hybrid cells in selective medium. In addition, methotrexate hybrids develop more rapidly and can therefore be processed earlier. The stability and low toxicity of methotrexate also favor its use in the selection of hybridomas.
Aminopterin↗
Hybridoma cell lines secreting monoclonal antibodies against equine infectious anemia virus.
A monoclonal anti-equine infectious anemia virus (anti-EIAV) antibody (1B15) has been generated by fusion of X63 Ag 8.653 myeloma cells and spleen cells from mice hypersensitized with viral antigen p29. Ouchterlony double-diffusion analysis indicated that antibody 1B15 is of the IgG class. The specificity of the immune reaction for p29 was confirmed by cross-over immunoelectrophoresis and disc-gel electrophoresis. MAb 1B15 was used to devise a solid-phase 'capture' RIA for EIAV-p29 antigen. The antigen, bound by 1B15 adsorbed onto wells of flexible microtitre plates, was detected using a rabbit anti-p29 serum followed by a 125I-labelled tracer. The assay was applied to detected using a of virus in horse serum and infected cell culture fluids.
Animals↗
Monoclonal antibodies specific for carcinoembryonic antigen produced by hybridoma technology.
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Animals↗
Rapid solid-phase radioimmunoassay for detection of equine infectious anemia viral antigen and antibodies: parameters involved in standardization.
Solid-phase radioimmunoassays (SPRIA) are described for the detection of equine infectious anemia (EIA) viral antigen and antibodies. Protein-antigen P29 currently used in the agar-gel immunodiffusion (AGID) test was used as antigen in the SPRIA. Rabbit sera selected from positive AGID test data were used to standardize the method. Briefly, wells of flexible microtitre plates coated with antigen were incubated with antiserum followed by a secondary labelled antibody. The radioactivity remaining in the wells after washing provided a measure of the amount of specific antibodies in the serum. When testing a group of rabbit sera, negative for EIA virus antibodies by the AGID test, in the SPRIA a range of positive reactivities was noted. The specificity of the reaction was assessed by inhibition with the antigen. The reaction of immune serum against EIA-virus antigen adsorbed to the wells, was completely inhibited by the antigen in solution. This property was applied in an indirect competitive SPRIA for the detection of viral protein P29. The detection threshold of the SPRIA for EIA virus protein was about 5 ng and about 1 ng of antibody can be detected. The assay is rapid, specific and sensitive and allows the testing of multiple serum samples with the advantage of employing a single secondary labelled antibody.
Animals↗
Human lymphocyte capping and the effect of lanthanum in Duchenne muscular dystrophy.
Capping was studied in lymphocytes obtained from controls and patients with Duchenne muscular dystrophy. Before the induction of capping by anti-Ig antibodies the cells were incubated with increasing concentrations of calcium and lanthanum. It was found that the presence or absence of extracellular calcium had no effect. Lanthanum, which blocks active calcium extrusion, altered the proportion of capping in both controls and Duchenne lymphocytes. These observations are discussed in relation to the possible involvement of free intracellular calcium in normal controls and patients with Duchenne muscular dystrophy.
Calcium↗
Human lymphocyte capping in Duchenne muscular dystrophy.
We have confirmed the observation that, in patients with Duchenne muscular dystrophy, there is a significant reduction in lymphocyte capping. However, our results indicated that this may be related to the degree of muscle damage in these boys, since it was correlated to both age and serum creatine kinase activity and could therefore be a secondary phenomenon.
Adolescent↗